Single-cell Analysis of Glycomic and Proteomic Features of the HIV Reservoir

HIV 病毒库的糖组学和蛋白质组学特征的单细胞分析

• 批准号: 10672296
• 负责人: Mohamed Abdel Mohsen
• 金额: $ 23.12万
• 依托单位: WISTAR INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10672296
• 关键词: Abnormal Cell; Atlases; Back; Binding; Bioinformatics; Biological Markers; Biology; Blood; CD4 Positive T Lymphocytes; Carbohydrates; Cell Separation; Cell Surface Proteins; Cell physiology; Cell surface; Cells; DNA; Data; Development; Disease; Frequencies; Genetic Transcription; Genome; Goals; HIV; HIV Infections; HIV antiretroviral; HIV therapy; Immunologics; In Vitro; Individual; Interruption; Link; Lymphoid Tissue; Maps; Membrane Proteins; Modeling; Patient observation; Patients; Pattern; Peripheral; Persons; Phenotype; Play; Polysaccharides; Process; Proteins; Proteomics; Proviruses; Reporter; Role; Sorting; Specimen; Surface; T-Cell Activation; Techniques; Technology; Testing; Therapeutically Targetable; Transcript; Validation; Viral Proteins; antiretroviral therapy; biomarker identification; clinically relevant; design; glycosylation; immune function; in vivo; insight; novel; novel marker; single cell analysis; specific biomarkers; sugar; therapeutic target

PROJECT SUMMARY: It has emerged in recent years that multiple types of HIV reservoir cells can persist in the face of antiretroviral therapy (ART). HIV reservoir cells include those that are “latent” (transcriptionally- inactive) and those that are transcriptionally-active. The transcriptionally-active reservoir cells may be a major contributor to HIV rebound after ART interruption, as shown in analytic treatment interruption studies. In addition, some reservoir cells are able to produce viral proteins upon stimulation (i.e., inducible reservoir cells), and these reservoir cells may also be important for rebound as they are enriched for genome-intact and replication- competent HIV provirus. Unfortunately, the lack of a detailed understanding of the cell surface phenotypes of these HIV reservoir cells has precluded a full understanding of the biology of HIV persistence and hampered the development of a cure. Importantly, prior efforts to characterize reservoir phenotypes all focused on cell surface proteins and ignored cell surface glycans, despite the fact that glycans play a critical role in regulating multiple key cellular processes and immunological functions and have served as useful biomarkers in many diseases. We recently demonstrated that peripheral CD4+ T cells harboring high levels of fucosylated carbohydrates are enriched for HIV+ transcriptionally-active cells and deficient in HIV+ transcriptionally-inactive ones, and that this fucosylation is directly induced by HIV infection. These studies suggest that glycans may have utility as novel biomarkers of specific types of reservoir cells. In this study, we combine newly developed single-cell surface glycomic + proteomic technologies with bioinformatic analyses to analyze cells from an HIV latency model and ART-suppressed HIV+ individuals in order better understanding the makeup of these cells, with the ultimate goal of understand how they can persist in the face of ART and whether they express a unique profile of therapeutically targetable biomarkers. In Aim 1, we will test the hypothesis that cell-surface glycosylation patterns, including fucosylation, distinguish transcriptionally-active and inactive HIV reservoir cells in vitro and in vivo. These studies will take advantage of our recently established technique CyTOF-Lec, which pairs in-depth CyTOF phenotyping with surface glycan characterization at the single-cell level. In Aim 2, we will examine the glycan features of in vivo inducible reservoir cells. To do this, we will adapt our recently established and validated PP-SLIDE (predicted precursor single-cell linkage using distance estimation) approach to characterize the glycan + protein features of patient-derived reservoir cells inducible by ex vivo stimulation. Together, these analyses will allow us to test our central hypothesis that specific cell-surface glycosylation patterns, including fucosylation, can distinguish HIV reservoir cells from uninfected ones, and furthermore distinguish different types of reservoir cells (e.g., latent, transcriptionally-active, and inducible).

项目摘要：近年来已经出现了多种类型的HIV储层细胞可以持续存在 抗逆转录病毒疗法的面孔（ART）。 HIV储层细胞包括那些“潜伏”的细胞（转录 - 不活动的）和转录活性的。转录活性储层细胞可能是主要的 如分析治疗中断研究所示，在ART中断后艾滋病毒反弹的贡献者。此外， 一些储液细胞能够在刺激时产生病毒蛋白（即诱导液储层细胞），并且这些细胞可以 储层细胞对反弹也可能很重要，因为它们富含基因组直觉和复制 - 有能力的HIV病毒。不幸的是，缺乏对细胞表面表型的详细理解 这些HIV储层细胞已经完全理解了对HIV持久性的生物学，并阻碍了 治愈的开发。重要的是，先前努力表征储层表型都集中在细胞表面上 蛋白质并忽略了细胞表面聚糖，使法在调节多重方面起着至关重要的作用。 关键的细胞过程和免疫功能，并在许多疾病中充当有用的生物标志物。 我们最近证明了外围CD4+ T细胞具有高水平的岩藻糖基化碳液体是 富含HIV+转录活性细胞，缺乏HIV+转录活性细胞，这是 HIV感染直接诱导岩藻糖基化。这些研究表明，聚糖可能具有新颖的效用 特定类型的储层细胞的生物标志物。在这项研究中，我们结合了新开发的单细胞表面 具有生物信息学分析的糖基 +蛋白质组学技术，以分析来自HIV潜伏期模型的细胞 和艺术抑制的艾滋病毒+个人，以便更好地了解这些细胞的构成，并具有最终 了解他们如何在艺术面前坚持下去以及他们是否表达独特的形象的目标 具有治疗目标的生物标志物。 在AIM 1中，我们将检验以下假设：细胞表面糖基化模式，包括岩藻糖基化，区分 在体外和体内转录活性和无活跃的HIV储量细胞。这些研究将利用 我们最近建立的技术Cytof-Lec，该技术将深入的细胞分型与表面聚糖配对 在单细胞级别的表征。在AIM 2中，我们将检查体内可诱导液的聚糖特征 细胞。为此，我们将调整我们最近建立和验证的PP-Slide（预测的前体单细胞） 使用距离估计的链接）方法以表征患者衍生的聚糖 +蛋白质特征 通过体内刺激可诱导的储层细胞。 总之，这些分析将使我们能够测试特定细胞表面糖基化的中心假设 包括岩藻糖基化在内的模式可以将HIV储层细胞与未感染的细胞区分开，然后将其区分开 不同类型的储层细胞（例如潜在，转录活性和诱导）。